Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins, which enables regulation of the activity of mature proteins by altering their structure and function.
Protein phosphorylation plays a pivotal role in cellular signal transduction. Among the biological functions controlled by this type of postranslational modification are: cell division, differentiation, and death (apoptosis); cell motility and cytoskeletal structure; control of DNA replication, transcription, splicing and translation; protein translocation events from the endoplasmic reticulum and Golgi apparatus to the membrane and extracellular space; protein nuclear import and export; and regulation of metabolic reactions, etc. Abnormal protein phosphorylation is widely recognized to be causally linked to the etiology of many diseases including cancer as well as immunologic, neuronal and metabolic disorders.
The presence of a phosphate moiety can modulate protein function in multiple ways. For example, common mechanism includes alterations in the catalytic properties (Vmax and Km) of an enzyme, leading to its activation or inactivation. A second widely recognized mechanism involves promoting protein-protein interactions. One example is the tyrosine autophosphorylation of the ligand-activated EGF receptor tyrosine kinase. This event triggers high-affinity binding to the phosphotyrosine residue of the receptor's C-terminal intracellular domain to the SH2 motif of the adaptor molecule Grb2. Grb2, in turn, binds through its SH3 motif to a second adaptor molecule, such as SHC. The formation of this ternary complex activates the signaling events that are responsible for the biological effects of EGF. Serine and threonine phosphorylation events also have been recently recognized to exert their biological function through protein-protein interaction events that are mediated by the high-affinity binding of phosphoserine and phosphothreonine to WW motifs present in a large variety of proteins (Lu, P. J. et al (1999) Science 283:1325-1328).
A third important outcome of protein phosphorylation is changes in the subcellular localization of the substrate. As an example, nuclear import and export events in a large diversity of proteins are regulated by protein phosphorylation (Drier E. A. et al (1999) Genes Dev 13: 556-568).
Protein kinases are one of the largest families of eukaryotic proteins with several thousand known members. These proteins share a 250-300 amino acid domain that can be subdivided into 12 distinct subdomains that comprise the common catalytic core structure. These conserved protein motifs have recently been exploited using PCR-based and bioinformatic strategies leading to a significant expansion of the known kinases. Multiple alignment of the sequences in the catalytic domain of protein kinases and subsequent parsimony analysis permits their segregation into subfamilies of related kinases.
The best characterized protein kinases in eukaryotes phosphorylate proteins on the hydroxyl substituent of serine, threonine and tyrosine residues, which are the most common phospho-acceptor amino acid residues. However, phosphorylation on histidine has also been observed in bacteria.
Kinases fall largely into two groups: those specific for phosphorylating serines and threonines, and those specific for phosphorylating tyrosines. Some kinases, referred to as “dual specificity” kinases, are able to phosphorylate tyrosine as well as serine/threonine residues. Protein kinases can also be characterized by their location within the cell. Some kinases are transmembrane receptor-type proteins capable of directly altering their catalytic activity in response to the external environment, such as the binding of a ligand. Other kinases are non-receptor type proteins lacking any transmembrane domain; they can be found in a variety of cellular compartments from the inner surface of the cell membrane to the nucleus.
Many kinases are involved in regulatory cascades. The substrates of such kinases can include other kinases whose activities are regulated by their phosphorylation state. Ultimately, the activity of some downstream effector is modulated by phosphorylation resulting from activation of such a pathway. The conserved protein motifs of these kinases have recently been exploited using PCR-based cloning strategies leading to a significant expansion of the known kinases.
Multiple alignment of the sequences in the catalytic domain of protein kinases and subsequent parsimony analysis permits the segregation of related kinases into distinct branches of subfamilies including: tyrosine kinases (PTK's), dual-specificity kinases, and serine/threonine kinases (STK's). The latter subfamily includes cyclic nucleotide-dependent kinases, calcium/calmodulin kinases, cyclin-dependent kinases (CDKs), MAP kinases, serine-threonine kinase receptors, as well as and several other subfamilies.
The protein kinases can be classified into several major groups including AGC, CAMK, Casein kinase 1, CMGC, STE, tyrosine kinases, and atypical kinases (Plowman, G D et al., Proceedings of the National Academy of Sciences, USA, Vol. 96, Issue 24, 13603-13610, Nov. 23, 1999). In addition, there are a number of minor yet distinct families, including families related to worm- or fungal-specific kinases, and a family designated “other” to represent several smaller families. Within each group are several distinct families of more closely related kinases. In addition, an “atypical” family represents those protein kinases whose catalytic domain has little or no primary sequence homology to conventional kinases, including the A6 kinases and PI3 kinases.
The AGC kinases are basic amino acid-directed enzymes that phosphorylate residues found proximal to Arg and Lys. Examples within this group include the G protein-coupled receptor kinases (GRKs), the cyclic nucleotide-dependent kinases (PKA, PKC, PKG), NDR or DBF2 kinases, ribosomal S6 kinases, AKT kinases, myotonic dystrophy kinases (DMPKs), MAPK interacting kinases (NINKs), MAST kinases, and others.
The CAMK kinases (Ca2+/calmodulin-regulated kinases) are also basic amino acid-directed kinases. They include the Ca2+/calmodulin-regulated and AMP-dependent protein kinases (AMPK), myosin light chain kinases (MLCK), MAP kinase activating protein kinases (MAPKAPKs), checkpoint 2 kinases (CHK2), death-associated protein kinases (DAPKs), phosphorylase kinase (PHK), Rac and Rho-binding Trio kinases, a “unique” family of CAMKs, and the EMK-related protein kinases. The EMK family of STKs are involved in the control of cell polarity, microtubule stability and cancer. One member of the EMK family, C-TAK1, has been reported to control entry into mitosis by activating Cdc25C which in turn dephosphorylates Cdc2. Also included in the EMK family is MAKV, which has been shown to be overexpressed in metastatic tumors (Korobko IV, Kabishev A A, Kiselev S L. Dokl. Akad. Nauk 354 (4), 554-556 (1997)).
The CMGC kinases “proline-directed” enzymes that phosphorylate residues which exist in a proline-rich context. They include the cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinases-3 (GSK3s), RNA Dependent Helicase Kinase, and CDC-like kinase (CLK). Most CMGC kinases have larger than average kinase domains owing to the presence of insertions within subdomains X and XI. CDKs play a pivotal role in the regulation of mitosis during cell division. The process of cell division occurs in four stages: S phase, the period during which chromosomes duplicate, G2, mitosis (M), and G1 or interphase. During mitosis the duplicated chromosomes are evenly segregated allowing each daughter cell to receive a complete copy of the genome. A key mitotic regulator in all eukaryotic cells is the STK cdc2, a CDK regulated by cyclin B. However some CDK-like kinases, such as CDK5 are not cyclin associated, nor are they cell cycle regulated.
MAPKs play a pivotal role in many cellular signaling pathways, including stress response and mitogenesis (Lewis, T. S., Shapiro, P. S., and Ahn, N. G. (1998) Adv. Cancer Res. 74, 49-139). Growth factors such as EGF, and cytokines such as TNF-alpha, can activate MAP kinases. In response to EGF, Ras becomes activated and recruits Rafl to the membrane where Rafl is activated by mechanisms that can involve phosphorylation and conformational changes (Morrison, D. K., and Cutler, R. E. (1997) Curr. Opin. Cell Biol. 9, 174-179). Active Rafl phosphorylates MEK1 (MAP kinase kinase) which in turn phosphorylates and activates extracellular signal-regulated protein kinase (ERKs).
The tyrosine kinase group encompasses both cytoplasmic (e.g. src) as well as transmembrane receptor tyrosine kinases (e.g. EGF receptor). These kinases play a pivotal role in the signal transduction processes that mediate cell proliferation, differentiation, and apoptosis.
The STE family refers to the three classes of protein kinases that lie sequentially upstream of the MAPKs. This group includes STE7 (MEK or MAPKK) kinases, STE11 (MEKK or MAPKKK) kinases and STE20 (MEKKK) kinases. In humans, several protein kinase families that bear only distant homology with the STE11 family also operate at the level of MAPKKKs including RAF, MLK, TAK1, and COT. Since crosstalk takes place between protein kinases functioning at different levels of the MAPK cascade, the large number of STE family kinases could translate into an enormous potential for upstream signal specificity. The prototype, STE20 from baker's yeast, is regulated by hormone receptor signaling to directly affect cell cycle progression through the modulation of CDK activity. STE20 also coordinately regulates changes in the cytoskeleton and in transcriptional programs in a bifurcating pathway. In a similar manner, the homologous kinases in humans are likely to play a role in extracellular regulation of growth, cell adhesion and migration, and changes in transcriptional programs, all three of which have critical roles in tumorigenesis. Mammalian STE20-related protein kinases have been implicated in response to growth factors or cytokines, oxidative-, UV-, or irradiation-related stress pathways, inflammatory signals (e.g. TNFα), apoptotic stimuli (e.g. Fas), T and B cell costimulation, the control of cytoskeletal architecture, and cellular transformation.
Typically, the STE20-related kinases serve as upstream regulators of MAPK cascades. Examples include: HPK1, a protein-serine/threonine kinase (STK) having a STE20-like kinase domain that activates a protein kinase pathway leading to the stress-activated protein kinase, SAPK/JNK; PAK1, an STK with an upstream CDC42-binding domain that interacts with Rac and plays a role in cellular transformation through the Ras-MAPK pathway; and murine NIK, which interacts with upstream receptor tyrosine kinases and connects with downstream STE 11 family kinases.
NEK kinases are related to NIMA which is required for entry into mitosis in the filamentous fungus, A. nidulans. Mutations in the nimA gene cause the nim (“never in mitosis”) G2 arrest phenotype in this fungus (Fry, A. M. and Nigg, E. A. (1995) Current Biology 5: 1122-1125). Several observations suggest that higher eukaryotes can have a NIMA functional counterpart(s): (1) expression of a dominant-negative form of NIMA in HeLa cells causes a G2 arrest; (2) overexpression of NIMA causes chromatin condensation, not only in A. nidulans, but also in yeast, Xenopus oocytes and HeLa cells (Lu, K. P. and Hunter, T. (1995) Prog. Cell Cycle Res. 1, 187-205); (3) NIMA, when expressed in mammalian cells, interacts with pinl, a phosphorylation-specific prolyl isomerase that functions in cell cycle regulation (Lu, K. P. et al. (1996) Nature 380, 544-547); (4) okadaic acid inhibitor studies suggests the presence of cdc2-independent mechanism to induce mitosis (Ghosh, S. et al.(1998) Exp. Cell Res. 242, 1-9); and (5) a NIMA-like kinase (fin 1) exists in another eukaryote besides Aspergillus, Saccharomyces pombe (Krien, M. J. E. et al.(1998) J. Cell Sci. 111, 967-976).
Four mammalian NIMA-like kinases have been identified: NEK1, NEK2, NEK3 and NRK2. Despite the similarity of the mammalian NIMA-related kinases to NIMA over the catalytic region, the mammalian kinases are structurally different from NIMA over the extracatalytic regions. In addition, the mammalian kinases do not complement the nim phenotype in Aspergillus nimA mutants.
The casein kinase, CK1, family represents a distant branch of the protein kinase family. One or more forms are ubiquitously distributed in mammalian tissues and cell lines. CK1 kinases are found in cytoplasm, in nuclei, membrane-bound, and associated with the cytoskeleton. Splice variants differ in their subcellular distribution.
Several families cluster within a group of unrelated kinases termed “Other”. Included are: CHK1; Elongation 2 factor kinases (EIFK); homologues of the yeast sterile family kinases (STE), which refers to 3 classes of kinases which lie sequentially upstream of the MAPKs; Calcium-calmodulin kinase kinases (CAMKK); dual-specific tyrosine kinases (DYRK); IkB kinases (IKK); Integrin receptor kinase (IRAK); endoribonuclease-associated kinases (IRE); Mixed lineage kinase (MLK); LIM-domain containing kinase (LIMK); MOS; PIM; Receptor interacting kinase (RIP); SR-protein specific kinase (SRPK); RAF; Serine-threonine kinase receptors (STKR); TAK1; Testis specific kinase (TSK); tousled-related kinase (TSL); UNC51-related kinase (UNC); VRK; WEE; mitotic kinases (BUB 1, AURORA, PLK, and NIMA/NEK); several families that are close homologues to worm (C26C2.1, YQ09, ZC581.9, YFL033c, C24A1.3); Drosophila (SLOB), or yeast (YDOD sp, YGR262 sc) kinases; and others that are “unique,” and do not cluster into any obvious family. Additional families, first identified in lower eukaryotes such as yeast or worms, include YNL020, YPL236, YQ09, YWY3, SCY1, CO1H6.9, and C26C2.1.
RIP2 is a serine-threonine kinase associated with the tumor necrosis factor (TNF) receptor complex and is implicated in the activation of NF-kappa B and cell death in mammalian cells. It has recently been demonstrated that RIP2 activates the MAPK pathway (Navas, et al., J. Biol. Chem. 1999 Nov. 19; 274(47):33684-33690). RIP2 activates AP-1 and serum response element-regulated expression by inducing the activation of the Elk1 transcription factor. RIP2 directly phosphorylates and activates ERK2 in vivo and in vitro. RIP2, in turn, is activated through its interaction with Ras-activated Rafl. These results highlight the integrated nature of kinase signaling pathways.
The tousled (TSL) kinase was first identified in the plant Arabidopsis thaliana. TSL encodes a serine/threonine kinase that is essential for proper flower development. Human tousled-like kinases (Tlks) are cell-cycle-regulated enzymes, displaying maximal activities during S phase. This regulated activity suggests that Tlk function is linked to ongoing DNA replication (Sillje, et al., EMBO. 1999 Oct. 15;18(20):5691-5702).
The “histidine” kinases are abundant in prokaryotes, with more than 20 representatives in E. coli, and have also been identified in yeast, molds, and plants. In response to external stimuli, these kinases act as part of two-component systems to regulate DNA replication, cell division, and differentiation through phosphorylation of an aspartate in the target protein. To date, no “histidine” kinases have been identified in metazoans, although mitochondria pyruvate dehydrogenase kinase (PDK) and branched chain alpha-ketoacid dehydrogenase (BCKD) kinase, have sequence homology. PDK and BCKD kinases represent a unique family of atypical protein kinases involved in the regulation of glycolysis, the citric acid cycle, and protein synthesis during protein malnutrition. Structurally, they conserve only the C-terminal portion of “histidine” kinases including the G box regions. BCKD kinase phosphorylates the Ela subunit of the BCKD complex on Ser-293, proving it to be a functional protein kinase.
There are several proteins with protein kinase activity that appear to be structurally unrelated to the eukaryotic protein kinases. These include: Dictyostelium myosin heavy chain kinase A (MHCKA); Physarum polycephalum actin-fragmin kinase; the human A6 PTK; human BCR; mitochondria pyruvate dehydrogenase and branched chain fatty acid dehydrogenase kinase; and the prokaryotic “histidine” protein kinase family. The slime mold, worm, and human eEF-2 kinase homologues have all been demonstrated to have protein kinase activity, yet they bear little resemblance to conventional protein kinases except for the presence of an identified GxGxxG ATP binding motif.
Several other proteins contain protein kinase-like homology including: receptor guanylyl cyclases, diacylglycerol kinases, choline/ethanolamine kinases, and YLK1-related antibiotic resistance kinases. Each of these families contains short motifs that were recognized by profile searches with low scoring E-values, but a priori would not be expected to function as protein kinases. Instead, the similarity could simply reflect the modular nature of protein evolution and the primal role of ATP binding in diverse phosphotransfer enzymes. However, studies of a bacterial homologue of the YLK1 family suggests that the aminoglycoside phosphotransferases (APHs) are structurally and functionally related to protein kinases (Daigle, D. M., McKay, G. A., Thompson, P. R., Wright, G. D, Chem. Biol., 6(1):11-8, (1999)). There are over 40 APHs identified from bacteria that are resistant to aminoglycosides such as kanamycin, gentamycin, or amikacin. The crystal structure of one well characterized APH reveals that it shares greater than 40% structural identity with the 2 lobed structure of the catalytic domain of cAMP-dependent protein kinase (PKA), including an N-terminal lobe composed of a 5-stranded antiparallel beta sheet, and the core of the C-terminal lobe including several invariant segments found in all protein kinases. APHs lack the GxGxxG consensus sequence normally present in the loop between beta strands 1 and 2, but contain 7 of the 12 strictly conserved residues present in most protein kinases, including the HGDxxxN signature sequence in kinase subdomain VIB. Furthermore, APH also has been shown to exhibit protein serine/threonine kinase activity, suggesting that other YLK-related molecules can indeed be functional protein kinases.
The eukaryotic lipid kinases (PI3Ks, PI4Ks, and PIPKs) also contain several short motifs similar to protein kinases, but otherwise share minimal primary sequence similarity. However, once again, structural analysis of PIPKII-beta defines a conserved ATP-binding core that is strikingly similar to conventional protein kinases. Three residues are conserved among all of these enzymes including (relative to the PKA sequence) Lys-72 which binds the gamma phosphate of ATP, Asp-166, which is part of the HRDLK (SEQ ID NO:68) motif, and Asp-184 from the conserved Mg2+ or Mn2+-binding DFG motif. The worm genome contains 12 phosphatidylinositol kinases, including three PI3-kinases, two PI4-kinases, four PIP5-kinases, and 4 PI3-kinase-related kinases. The latter group has 4 mammalian members (DNA-PK, FRAP/TOR, ATM, and ATR), which have been shown to participate in the maintenance of genomic integrity in response to DNA damage, and exhibit true protein kinase activity, thus suggesting that other PI-kinases can also act as protein kinases. Regardless of whether they have true protein kinase activity, PI3-kinases are tightly linked to protein kinase signaling, as evidenced by their downstream involvement with many growth factor receptors and as upstream activators of the cell survival response mediated by the AKT protein kinase.
Human citron kinase is a serine-threonine protein kinase involved in cytokinesis and apoptosis. The mouse ortholog was described in citron-kinase knockout mice studies (Di Cunto, et al. Neuron 28:115-127, 2000) and proposed by in vitro studies to be a crucial effector of Rho in regulation of cytokinesis. In the study by Di Cunto, et al., citron-K knock-out mice grew at slower rates, were severly ataxic, and died before adulthood as a consequence of fatal seizures. Their brains displayed defective neurogenesis, with depleted specific neuronal populations. These types of abnormalities typically arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Thus, human citron kinase can be of great value in the treatment and prevention of central nervous system-related diseases, disorders, and conditions.
The following abbreviations are used to represent the kinases described throughout the present disclosure: CitK: Citron Kinase; DM: myotonic dystrophy kinase; PsPK5: Pisum sativum protein kinase 5; SGK1: Serum and glucocorticoid-regulated kinase; COT-1; COUP transcription factor 1 (also called COUP-TF1, COUP-TF I, and V-ERBA related protein EAR-3); MAST250: Microtubule-associated Kinase; ATPK5: Arabidopsis thaliana protein kinase 5; ATPK64: Arabidopsis thaliana Protein kinase 64; ATPK67: Arabidopsis thaliana protein kinase 67, and EGFR kinase: Epidermal Growth Factor receptor kinase.